Field of the Invention
The present invention relates to a method for quantifying a target nucleic acid sequence by use of a common internal control.
Description of the Related Art
A target nucleic acid amplification process is prevalently involved in most of technologies for detecting target nucleic acid sequences. Nucleic acid amplification is a pivotal process for a wide variety of methods in molecular biology, such that various amplification methods have been proposed.
The most predominant process for nucleic acid amplification known as polymerase chain reaction (hereinafter referred to as “PCR”) is based on repeated cycles of denaturation of double-stranded DNA, followed by oligonucleotide primer annealing to the DNA template, and primer extension by a DNA polymerase (Mullis et al. U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; Saiki et al., (1985) Science 230, 1350-1354).
As alternatives, various methods such as LCR (Ligase Chain Reaction), DA (Strand Displacement Amplification), NASBA (Nucleic Acid Sequence-Based Amplification), TMA (Transcription Mediated Amplification) and RCA (Rolling-Circle Amplification) have been suggested.
As application of nucleic acid amplification to target detection, the real-time detection methods are widely utilized to detect a target sequence with measuring nucleic acid amplification in a real-time manner. The real-time detection methods generally use labeled probes specifically hybridized with target sequences. As examples of methods using hybridization between labeled probes and target sequences, the Molecular beacon method using dual-labeled probes capable of hairpin structure (Tyagi et al, Nature Biotechnology v. 14 March 1996), the Hybridization probe method using two probes singly labeled with donor or acceptor (Bernad et al, 147-148 Clin Chem 2000; 46) and the Lux method using single-labeled oligonucleotides (U.S. Pat. No. 7,537,886) have been developed. In addition, the TaqMan method using cleavage reaction of a dual-labeled probe by the 5′-nuclease activity of DNA polymerases as well as hybridization of dual-labeled probes has been widely employed (U.S. Pat. Nos. 5,210,015 and 5,538,848). Other real-time detection methods is to use labeled primers, for example, including Sunrise primer method (Nazarenko et al, 2516-2521 Nucleic Acids Research, 1997, v. 25 no. 12, and U.S. Pat. No. 6,117,635), Scorpion primer method (Whitcombe et al, 804-807, Nature Biotechnology v. 17 Aug. 1999 and U.S. Pat. No. 6,326,145) and TSG primer method (WO 2011/078441).
As alternative approaches, real-time detection methods using duplexes formed depending on the presence of target nucleic acid sequences have been proposed: Invader assay (U.S. Pat. No. 5,691,142, U.S. Pat. No. 6,358,691 and U.S. Pat. No. 6,194,149), PTOCE (PTO cleavage AND extension) method (WO 2012/096523), PCE-SH (PTO Cleavage and Extension-Dependent Signaling Oligonucleotide Hybridization) method (WO 2013/115442), PCE-NH (PTO Cleavage and Extension-Dependent Non-Hybridization) method (PCT/KR2013/012312).
In the real-time detection methods as homogenous assay, the amplification and detection analysis are performed in a single tube such their performance is relatively convenient. In addition, the real-time detection methods are free from contaminations. As alternatives for target detection in a homogenous assay, the post-PCR melting assay has been suggested in which amplicons are detected using the melting profile (U.S. Pat. No. 5,871,908; U.S. Pat. No. 6,174,670, WO 2012/096523, WO 2013/115442 and PCT/KR2013/012312).
In the meantime, the quantification of target sequences as well as target detection is usually required for prognosis determination and analysis of drug responsiveness in the diagnostic field.
For quantification of target sequences, methods using signals (e.g., Ct value) and standard curve obtained by real-time PCR have been proposed (Bustin, J Mol Endocrinal 25:169 (2000), and Pfaffl and Hageleit, Biotechn Lett 23:275 (2001)). Although these quantification approaches are convenient, they have serious problems due to loss in sample extraction step and PCR inhibition.
In addition, there has been suggested a relative quantification method by real-time PCR in which expression level of a certain gene in an unknown sample is compared with that of the certain gene in a control sample. To calculate a relative expression ratio, methods using ΔCP (delta CP) or ΔΔCt (delta delta Ct) value with or without efficiency correction have been reported (Livak et al., Methods, 25(4):402 (2001), Souaze F et al., Biotechniques 21(2): 280 (1996) and Pfaffl M W, AZ of Quantitative PCR pp: 87120 (2004) La Jolla, Calif.: IUL Biotechnology Series, International University Line).
The ΔΔCt method measures not only expression level of a target gene but also expression level of a reference gene constitutively expressed in cells or tissues, and normalizes expression levels between samples by using the expression level of the reference gene. Generally, housekeeping genes such as the GAPDH gene and β-actin gene are used as the reference gene. They are likely to show different expression levels depending on cells (or tissues) or surrounding environments (reaction conditions and treatments). In this regard, the selection of housekeeping gene or reference gene suitable to analysis purpose and conditions is critical, which is time-consuming and troublesome work. To make matters worse, the selection may not be practical in some situations.
As approaches to detect and quantify target sequences by using PCR, a competitive PCR process using known-amount competitors having a primer binding site identical to that on target sequences has been proposed. While the competitor has a primer binding site identical to that on target sequences, it is differentially detected by difference in internal sequences or size of amplified products. For example, U.S. Pat. No. 5,213,961 discloses a competitive PCR process using gel electrophoresis. A competitive PCR process using post-PCR melting assay has been also proposed to quantify target sequences (Samiya Al-Robaiy et. et., BioTechniques 31:1382-1388 (2001)). These methods employ serially-diluted competitors for quantification, which are considered troublesome. As the competitors have a similar sequence to a target sequence for PCR efficiency, heteroduplexes are likely to form. Furthermore, the methods are compelled to use competitors with concentrations similar to those of unknown samples for preventing one of competitors and unknown samples to reach plateau.
Under such circumstances surrounding conventional technologies, there are long-felt needs to absolutely quantify target sequences in more convenient and rapid manner.
Throughout this application, various patents and publications are referenced and citations are provided in parentheses. The disclosure of these patents and publications in their entities are hereby incorporated by references into this application in order to more fully describe this invention and the state of the art to which this invention pertains.